Dülk Metta, Szeder Bálint, Glatz Gábor, Merő Balázs L, Koprivanacz Kitti, Kudlik Gyöngyi, Vas Virág, Sipeki Szabolcs, Cserkaszky Anna, Radnai László, Buday László
Institute of Enzymology, Research Centre for Natural Sciences , Hungarian Academy of Sciences , 1117 Budapest , Hungary.
Department of Anatomy, Cell and Developmental Biology , Eötvös Loránd University , 1117 Budapest , Hungary.
Biochemistry. 2018 Jul 17;57(28):4186-4196. doi: 10.1021/acs.biochem.8b00084. Epub 2018 Jul 5.
The nonreceptor tyrosine kinase Src is a central component of the epidermal growth factor (EGF) signaling pathway. Our group recently showed that the Frank-ter Haar syndrome protein Tks4 (tyrosine kinase substrate with four Src homology 3 domains) is also involved in EGF signaling. Here we demonstrate that Tks4 and Src bind directly to each other and elucidate the details of the molecular mechanism of this complex formation. Results of GST pull-down and fluorescence polarization assays show that both a proline-rich SH3 binding motif (PSRPLPDAP, residues 466-474) and an adjacent phosphotyrosine-containing SH2 binding motif (pYEEI, residues 508-511) in Tks4 are responsible for Src binding. These motifs interact with the SH3 and SH2 domains of Src, respectively, leading to a synergistic enhancement of binding strength and a highly stable, "bidentate"-type of interaction. In agreement with these results, we found that the association of Src with Tks4 is permanent and the complex lasts at least 3 h in living cells. We conclude that the interaction of Tks4 with Src may result in the long term stabilization of the kinase in its active conformation, leading to prolonged Src activity following EGF stimulation.
非受体酪氨酸激酶Src是表皮生长因子（EGF）信号通路的核心组成部分。我们团队最近发现，弗兰克 - 特哈尔综合征蛋白Tks4（具有四个Src同源结构域的酪氨酸激酶底物）也参与EGF信号传导。在此，我们证明Tks4和Src直接相互结合，并阐明了这种复合物形成的分子机制细节。谷胱甘肽 - S - 转移酶（GST）下拉实验和荧光偏振分析结果表明，Tks4中富含脯氨酸的SH3结合基序（PSRPLPDAP，第466 - 474位氨基酸残基）和相邻的含磷酸酪氨酸的SH2结合基序（pYEEI，第508 - 511位氨基酸残基）均负责与Src结合。这些基序分别与Src的SH3和SH2结构域相互作用，导致结合强度协同增强以及形成高度稳定的“双齿”型相互作用。与这些结果一致，我们发现Src与Tks4的结合是永久性的，并且该复合物在活细胞中至少持续3小时。我们得出结论，Tks4与Src的相互作用可能导致激酶在其活性构象中长期稳定，从而在EGF刺激后延长Src的活性。
